Fungal Genet Biol 48:15–22PubMed Bungihan ME, Tan MA, Kitajima M, Kogure N, Franzblau SG, dela Cruz TEE, Takayama H, Nonato MG (2011) Bioactive metabolites of Diaporthe sp. P133, an endophytic fungus isolated from Pandanus amaryllifolius. J Nat Med 65:606–609PubMed Burns E, Ifrach I, Carmeli S, Pawlik JR, Ilan M (2003) Comparison of antipredatory sedimentary lipids? Org Geochem 30:1–14 Cheng MJ, Wua MD, Yuan GF, Chen YL, Su YS, Hsieh MT, Chen IS (2012) Secondary metabolites and cytotoxic activities from

the endophytic fungus Annulohypoxylon squamulosum. Phytochem Lett 5:219–223 Cichewicz RH (2010) Epigenome manipulation as a pathway Opaganib to new natural product scaffolds and their congeners. Nat Prod Rep 27:11–22PubMed Cichewicz RH, Clifford LJ, Lassen PR, Cao X, Freedman TB, Nafie LA,

PI3K inhibitor Deschamps JD, Kenyon VA, Flanary JR, Holman TR, Crews P (2005) Stereochemical determination and bioactivity assessment of (S)-(+)-curcuphenol dimers isolated from the marine sponge Didiscus aceratus and synthesized through laccase biocatalysis. Bioorg Med Chem 13:5600–5612PubMed Cohen E, Koch L, Thu KM, Rahamim Y, Aluma Y, Ilan M, Yarden O, Carmeli S (2011) Novel terpenoids of the fungus Aspergillus insuetus isolated from the Mediterranean sponge Psammocinia sp. collected along the coast of Israel. Bioorg Med Chem 19:6587–6593PubMed Córdoba-Pedregosa MC, Villalba JM, Córdoba F, González-Reyes JA (2005) Changes in intracellular and apoplastic peroxidise activity, ascorbate redox status and root elongation induced by enhanced ascorbate content in Allium cepa L. J Exp Bot 56:685–694 Criddle RS, Hansen LD, Breidenbach RW, Ward MR, Huffaker RC (1989) Effects of NaCl on metabolic heat evolution rates by barley roots. Plant Physiol 90:53–58PubMed Debbab

A, Aly AH, Edrada-Ebel RA, Wray V, Müller WEG, Totzke F, Zirrgiebel U, Schächtele C, Kubbutat MHG, Lin WH, Mosaddak M, Hakiki A, Proksch P, Ebel R (2009) Bioactive metabolites from endophytic fungus Stemphylium globuliferum isolated from Mentha pulegium. J Nat Prod Quisqualic acid 72:626–631PubMed Debbab A, Aly AH, Lin WH, Proksch P (2010) Bioactive compounds from marine bacteria and fungi. Microbiol Biotechnol 3:544–563 Debbab A, Aly AH, Proksch P (2011) Bioactive secondary metabolites from terrestrial endophytes and associated marine derived fungi. Fungal Divers 49:1–12 Debbab A, Aly AH, Edrada-Ebel R, Wray V, Pretsch A, Pescitelli G, Kurtan T, Proksch P (2012) New anthracene derivatives—structure elucidation and antimicrobial activity. Eur J Org Chem 1351–1359. Ding B, Yin Y, Zhang F, Li Z (2011) Recovery and phylogenetic diversity of culturable fungi associated with marine sponges Clathrina luteoculcitella and Holoxea sp. in the South China Sea. Mar Biotechnol 13:713–721PubMed Ding G, Hl W, Chen L, Chen AJ, Lan J, Chen XD, Zhang HW, Chen H, Liu XZ, Zou ZM (2012) Cytochalasans with different amino-acid origin from the plant endophytic fungus Trichoderma gamsii.

princeps, which have lost the regulatory ‘ATC’ domain, or the loss of the ‘HTH’ domain of birA, the ‘PNPase C’ domain of rne and the ‘DEAD box A’ of dead in the case of M. endobia. Additionally, many other genes have been shortened due to frameshifts or the presence of premature stop codons, in comparison with their orthologs in free-living relatives (e.g. sspB, rplQ, rplO and aroC in T. princeps; thiC, ybgI, yacG, ygbQ, ftsL, ftsY and tilS in M. endobia). In some cases, the shortening removes some non-essential protein domains completely (e.g., engA, rpoA and rpoD in T. princeps; secA, aceF, yebA and metG in M. endobia). The loss of the ‘anticodon

binding domain of tRNA’ and ‘putative tRNA binding domain’ of metG, encoding methionyl-tRNA synthetase is common to other endosymbionts with reduced genomes. Finally, even though both genomes have an unusually high G + C content compared Tamoxifen in vivo with most bacterial endosymbionts, at least M. endobia seems to be suffering the AT mutational bias typical of bacterial genomes [27, 28]. This conclusion is drawn from the analysis of the nucleotide composition of genes, pseudogenes and IGRs (Table 1), as well as the preferential use of AT-rich codons (Additional file 2) including a high incidence of the TAA

stop codon (56.44%). Since both genomes seem to rely on the DNA replication and repair machinery of M. endobia (see next section), both genomes could be expected see more Cobimetinib cell line to undergo a similar trend towards an increase in AT content. However, this trend is undetectable in T. princeps, where the G + C content of pseudogenes and IGRs do not differ from that of the genes (Table 1). The differences in G + C content between both genomes could be due to a higher ancestral G + C content plus a slower

evolutionary rate for T. princeps, due to its extreme genome reduction, and the biology of the system (i.e., a lower replication rate, since each T. princeps cell retains several M. endobia cells). In fact, the codon usage bias (Additional file 2) and differences in the amino acidic composition between both endosymbiont proteomes (Figure 2) reflect their differences in G + C content. Thus, T. princeps proteins are rich in amino acids encoded by GC-rich codons (Ala, Arg, Leu, Gly, Val and Ser represent 56.82% of the total, whereas Phe and Trp are scarce), while M. endobia has a weaker amino acid composition bias (Additional file 2). Figure 2 Amino acid content profiles for T. princeps and M. endobia proteomes. Amino acids are ranked from left to right according to the GC-richness of the corresponding codons (see Additional data file 2). T. princeps genome comparison The genome alignment of both T. princeps strains showed a high degree of identity at the sequence level (99.98%, being 138,903 bp identical), which is coherent with their evolutionary proximity and extreme genome reduction.

Finite element simulations generally reproduced the experimental phonon and magnon dispersion relations. Because of the possibility of simultaneously controlling and manipulating the magnon and phonon propagation in them, magphonic crystals could find applications

in areas such as acoustic and spin-wave signal processing. Acknowledgment Financial support from the Ministry of Education, Singapore under grant R144-000-282-112 is gratefully acknowledged. References 1. Rolland Q, Oudich M, El-Jallal S, Dupont S, Pennec Y, Gazalet J, Kastelik JC, Leveque G, Djafari-Rouhani B: Acousto-optic couplings in two-dimensional phoxonic crystal cavities. Maraviroc Appl Phys Lett 2012, 101:061109.CrossRef 2. Laude V, Beugnot J-C, Benchabane S, Pennec Y, Djafari-Rouhani B, Papanikolaou N, Escalante JM, Martinez A: Simultaneous guidance of slow photons and slow acoustic phonons

in silicon phoxonic crystal slabs. Opt Express 2011, 19:9690–9698.CrossRef 3. El Hassouani Y, Li C, Pennec Y, El Boudouti EH, Larabi H, Akjouj A, Bou Matar O, Laude V, Papanikolaou N, Martinez A, Djafari Rouhani B: Dual phononic and photonic band gaps in a periodic array of pillars deposited on a thin plate. Phys Rev B 2010, 82:155405.CrossRef 4. Papanikolaou N, Psarobas IE, Stefanou R788 research buy N: Absolute spectral gaps for infrared light and hypersound in three-dimensional metallodielectric phoxonic crystals. Appl Phys Lett 2010, 96:231917.CrossRef 5. Nikitov S, Gulyaev Y, Grigorevsky V, Grigorevsky A, Lisenkov I, Popov R: Review of phononic crystals, nonlinear processes, devices and prospects. J Acoust Soc Am 2008, 123:3040.CrossRef 6. Zhang VL, Hou CG, Pan HH, Ma FS, Kuok MH, Lim HS, Ng SC, Cottam MG, Jamali M, Yang H: Phononic dispersion of a two-dimensional tuclazepam chessboard-patterned bicomponent array on a substrate. Appl Phys Lett 2012, 101:053102.CrossRef 7. Zhang VL, Ma FS, Pan HH, Lin CS, Lim HS, Ng SC, Kuok MH, Jain S, Adeyeye

AO: Observation of dual magnonic and phononic bandgaps in bi-component nanostructured crystals. Appl Phys Lett 2012, 100:163118.CrossRef 8. Kushwaha MS, Halevi P, Dobrzynski L, Djafari-Rouhani B: Acoustic band structure of periodic elastic composites. Phys Rev Lett 1993, 71:2022–2025.CrossRef 9. Cheng W, Wang J, Jonas U, Fytas G, Stefanou N: Observation and tuning of hypersonic bandgaps in colloidal crystals. Nat Mater 2006, 5:830–836.CrossRef 10. Wang ZK, Zhang VL, Lim HS, Ng SC, Kuok MH, Jain S, Adeyeye AO: Observation of frequency band gaps in a one-dimensional nanostructured magnonic crystal. Appl Phys Lett 2009, 94:083112.CrossRef 11. Jorzick J, Demokritov SO, Mathieu C, Hillebrands B, Bartenlian B, Chappert C, Rousseaux F, Slavin AN: Brillouin light scattering from quantized spin waves in micron-size magnetic wires. Phys Rev B 1999, 60:15194–15200.CrossRef 12.

As a well-known material used for

photographic film, AgCl Tyrosine Kinase Inhibitor Library high throughput has shown its valuable applications as visible light photocatalysts [2–8]. AgCl is a stable photosensitive semiconductor material with a direct band gap of 5.15 eV and an indirect band gap of 3.25 eV. Although the intrinsic light response of AgCl is located in the ultraviolet region as well, once AgCl absorbs a photon, an electron-hole pair will be generated and subsequently, the photogenerated electron combines with an Ag+ ion to form an Ag atom [7]. Finally, a lot of silver atoms are formed on the surface of the AgCl, which could extend the light response of AgCl into the visible light region [1, 6, 7]. Besides, the morphology of AgCl has significant influence on its photocatalytic activity, so it is important to develop facile methods to synthesize size- and shape-controlled AgCl materials. Recently, the facile hydrothermal method is employed to synthesize variable micro-/nano-AgCl structures, including AgCl nanocubes [6], cube-like Ag@AgCl [7], and even near-spherical AgCl crystal by an ionic liquid-assisted hydrothermal

method [8]. However, for AgCl microcrystals, this narrow morphology variation (simply varied from near-spherical to cubical [2–7]) inspired that more particular attention Deforolimus datasheet is deserved to pay on the novel AgCl morphologies, including the synthesis Methisazone methods and their generation mechanisms, even the possible morphology evolution

processes. Herein, the novel flower-like AgCl microstructures similar to PbS crystals [9] are synthesized by a facile hydrothermal process without any catalysts or templates. Also, a series of AgCl morphology evolution processes are observed. Flower-like structures are recrystallized after the dendritic crystals are fragmentized, assembled, and dissolved. The detailed mechanism of these evolution processes has been further discussed systemically. Furthermore, flower-like AgCl microstructures exhibited enhanced photocatalytic degradation of methyl orange under visible light. Methods The AgCl dendritic and flower-like structure are synthesized via hydrothermal method by reacting silver nitrate (AgNO3, 99.8%) with ethylene glycol (EG, 99%) in the presence of poly(vinyl pyrrolidone) (PVP-K30, MW = 30,000). In a typical synthesis, all the solutions are under constant stirring. Firstly, a 10-ml EG solution with 0.2 g of PVP was prepared. Then using droppers, another 7 ml of EG which contained 10 mM of AgNO3 is added. Afterwards, 3 ml of undiluted hydrochloric acid (HCl, 36% ~ 38%) is added into this mixture. The mixed AgNO3/ PVP/HCl/EG solution is further stirred for several minutes until it becomes uniform. This solution is then transferred into a 25-ml Teflon-lined autoclave tube and dried in the drying tunnel at 160°C for different times.

nucleatum to generate energy and produce ammonia, acetate and butanoate as end-products [45–47]. The bacterium also ferments sugars (glucose, galactose and fructose) to produce a mixture of acetate, formate and lactate [48]. Figure 2 Representation of protein groups that were regulated at pH 8.2 compared to 7.4. In the present study, key enzymes involved in this website the catabolism of glutamate and histidine via the 2-oxoglutarate pathway and pyruvate were significantly altered in biofilm cells (Table 1). A previous study of F. nucleatum

cultured at pH 6.4, 7.4 and 7.8 also revealed the regulation of metabolic enzymes [26]. In contrast to this finding, we found that no glycolytic enzyme concentrations were altered in biofilm cells grown at pH 8.2 compared to planktonic cells grown at 7.4. However, a three-fold increase in glucose utilisation and IP was observed (Table 2, Figure 3).

It is possible that the observed increase in glucose storage may play an important role in the organism’s survival during periods of nutrient limitation when exposed to pH 8.2 [43, 49, 50]. Although the expression of glycolytic enzymes was not significantly altered, an increase in lactate dehydrogenase (LDH) (EC 1.1.2.8) and a three-fold increase in lactate production was observed, indicating a metabolic Temsirolimus mw shift at pH 8.2 towards ATP generation via anaerobic glycolysis (Embden-Meyerhof-Parnas pathway) (Tables 1 and 2, Figure 3). In addition, at pH 8.2, an increase in acidic PIK3C2G end products per mg cellular protein and shift to lactate

production was observed (Table 2). These changes may assist in maintenance of intracellular pH due to the lower pKa of lactic acid (3.08) compared to formic (3.75), acetic (4.75) and butanoic (4.82) acids. . Table 2 Glucose consumption and metabolic end-products produced by F. nucleatum grown at pH 8.2 and 7.4 Growth pH Glucose utilisation1 IP2 Acidic end-products3 GDH4       Lactate Formate Acetate Butanoate   7.4 ± 0.1 23.1 ± 2.1 2.39 ± 0.12 5.7 ± 0.5 92.4 ± 8.6 59.4 ± 6.5 63.0 ± 5.1 8.87 ± 0.40 8.2 ± 0.1 65.9 ± 7.2 7.62 ± 0.71 18.3 ± 1.9 131.2 ± 11.6 115.3 ± 12.7 99.6 ± 10.8 13.73 ± 1.25 1Glucose utilisation expressed as mmoles of glucose g-1 cell protein. 2Intracellular polyglucose expressed as μg glucose mg-1 cell protein. 3Acidic end-products expressed in mmol g-1 cell protein. 4NAD-specific glutamate dehydrogenase (GDH) activity measured in cells expressed as GDH unit mg-1 cell protein Figure 3 Pathways for glucose and histidine/glutamate catabolism in F. nucleatum. Significantly regulated enzymes detected in this study at pH 8.2 are indicated by the enzyme commission (E.C) numbers (Refer to Table 1). Bold arrows indicate increased enzyme levels while double-slash indicates decreased enzyme expression.

brasiliensis. The nucleotide and amino acid positions are marked on the left side. Lower case letters represent the untranslated 5′ region. Bold letters in nucleotide sequence represent the start and stop codons. Two introns were found in the genomic sequence

and are shown in italic. Three conserved residues (marked with arrows) of amino acids (asparagine – D; histidine – H and serine – S) belonging to the active site of serine proteases from the subtilase family S08 are evidenced. Six putative RG-7204 N-glycosylation sites are marked in bold letters. A signal peptide formed by the first 16 amino acids is underlined. The TATA box in the promoter region is evidenced by white letters. A GATA binding region of the transcription factor AreA was found and is evidenced by a white box. (JPEG 270 KB) References 1. Rawlings ND, Barrett AJ: Evolutionary families of peptidases. Biochem J 1993, 290:205–218.PubMed 2. Jousson O, Lechenne B, Bontems O, Mignon B, Reichard U, Barblan J, Quadroni M, Monod M: Secreted subtilisin gene family in Trichophyton rubrum . Gene 2004, 339:79–88.PubMedCrossRef 3. Baldo A, Tabart J, Vermout S, Mathy A,

Collard A, Losson B, Mignon B: Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes. J Med Microbiol 2008, 57:1152–1156.PubMedCrossRef 4. Donofrio NM, Oh Y, Lundy R, Pan H, Brown DE, Jeong JS, Coughlan S, Mitchell TK, Dean RA: Global gene expression during nitrogen ABT-263 manufacturer starvation in the rice blast fungus, Magnaporthe grisea . Fungal Genet Biol 2006, 43:605–617.PubMedCrossRef 5. Wang B, Liu X, Wu W, Liu X, Li S: Purification, characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis . Microbiol Res 2009, 164:665–673.PubMedCrossRef 6.

Zou CG, Tao N, Molecular motor Liu WJ, Yang JK, Huang XW, Liu XY, Tu HH, Gan ZW, Zhang KQ: Regulation of subtilisin-like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea . Environ Microbiol 2010. 7. Franco M, Peracoli MT, Soares A, Montenegro R, Mendes RP, Meira DA: Host-parasite relationship in paracoccidioidomycosis. Curr Top Med Mycol 1993, 5:115–149.PubMed 8. Parente JA, Costa M, Pereira M, Soares CMA: Transcriptome overview of Paracoccidioides brasiliensis proteases. Genet Mol Res 2005, 4:358–371.PubMed 9. Carmona AK, Puccia R, Oliveira MC, Rodrigues EG, Juliano L, Travassos LR: Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis . Biochem J 1995, 309:209–214.PubMed 10. Puccia R, Carmona AK, Gesztesi JL, Juliano L, Travassos LR: Exocellular proteolytic activity of Paracoccidioides brasiliensis : cleavage of components associated with the basement membrane. Med Mycol 1998, 36:345–348.PubMed 11.

This would lead to symptoms such as shortness of breath, heart palpitations and fatigue [55]. Furthermore, when subjects were asked regarding the use of amino acid supplementation, all of them denied intake. Amino acid supplementation is not recommended for the fencers due to their high protein diet intake. These preliminary findings in lipid-lipoprotein profiles, in conjunction with the findings of unbalanced diet consumption among fencing players, demonstrate the need

for further research in this group of athletes. The results of several studies confirmed that saturated fatty acids leads to early development of CHD whereas monounsaturated and polyunsaturated fatty acids, significantly prevents the possibility of CHD [56–61]. The intake of monounsaturated Decitabine molecular weight fats and polyunsaturated fats were higher than the recommended values indicating appropriate choice of food yet, the diet consumption of the fencers is still high in total fat content when compared to the RDA values. Although selleck products the blood lipids profile test revealed Kuwaiti fencers have normal blood lipids, the dietary intake analysis showed an unbalanced macronutrients and micronutrients consumption. A dietary intervention for Kuwaiti fencers by qualified and registered

dietitians is needed to focus on healthy food choices and reduction of saturated fats. Reduced fiber intakes have many health complications. The subjects in the present study have very low intake of fiber in comparison with the value recommended by all diet agencies. The low fiber intake could cause certain types

of cancer and is associated with constipation, risk of heart disease and other digestive problems [62, 63]. The players consumed both calcium (Ca) and potassium (K) that were marginal in comparison with recommended values, therefore, the mineral content of the foods consumed was adequate for the athlete. However, it is important to avoid any deficiencies in Ca and K. Calcium, builds bones and prevents osteoporosis. Potassium, helps muscles and nerves function properly, maintains the proper electrolyte balance, acid-base Thiamet G balance and lowers the risk of hypertension [1]. The high quantity of sodium consumed by fencers (5306.6 ± 1033.9) exceeds the recommended by RDA (2300 mg/d). This is mostly due to the nature of the Kuwaiti diet and high percentage of fast food consumption. The current recommendation is to consume less than 2,400 milligrams (mg) of sodium a day. This is about one teaspoon of table salt per day. It includes all salt and sodium consumed, including sodium used in cooking and at the table. Although caffeine increases athletic performance and concentration it has adverse effects including possible anxiety, dependency, and withdrawal from the central nervous system [64–66].

For large-scale synthesis of nanoparticles in bioreactors, filamentous fungi are better agents for biomass production in comparison to algae and bacteria, since fungal mycelial mat can withstand flow pressure, agitation, and other conditions in the bioreactors [12]. Extracellular secretion of reductive proteins aids in extracellular synthesis of silver nanoparticles avoiding unnecessary cellular interference, and click here therefore, it is suitable for direct use in various applications. There are reports of mycosynthesis of silver nanoparticles using phytopathogenic fungi like Fusarium acuminatum [13], Aspergillus flavus [14], Alternaria

alternata [15], Coriolus versicolor [16], Penicillium fellutanum [17], and Fusarium semitectum [18]. Some fungi investigated were found to be capable of both extra- and intracellular biosynthesis of Ag-NPs having different particle sizes and shapes, but extracellular production of nanoparticles is more desirable from the point of view of easy isolation. Nanoparticles have some unique size- and shape-dependent physical and optical properties [19]. These unique characters are often responsible for their toxicity to various kinds of microbes such as bacteria, fungi, and also cancerous cells [20–22]. Hence, studies are going on regarding

their utility in the diagnosis as well as treatment of different kinds of diseases [23, 24]. In this regard, the presence of protein capping material is advantageous because this acts as the anchoring ever layer for drug or genetic materials FK506 concentration to be transported into human cells [25]. The presence of a nontoxic protein cap also increases uptake and retention inside human cells [26]. The present study deals with the extracellular biosynthesis of silver nanoparticles, using

cell-free extract of phytopathogenic soil-borne fungus Macrophomina phaseolina (Tassi) Goid, the causal organism of charcoal rot disease of about 500 agronomical important crops all over the world [27]. It describes not only a new method of green synthesis of silver nanoparticles but also their physical attributes, antibacterial activity against human and plant pathogenic multidrug-resistant bacteria, the inhibitory effect on the growth kinetics of microbes, the capping material around the silver nanoparticles, as well as their genotoxic effect. Methods M. phaseolina was grown in PDA medium at 28°C and was used for the synthesis of silver nanoparticles. The mycelium from solid substrate was inoculated in 50 ml potato dextrose broth (PDB) in 250-ml Erlenmeyer flasks and incubated at 28°C for 5 days. The fully expanded mycelial mat was harvested aseptically and washed with sterile distilled water to remove media components.

RT-PCR analysis of RNA extracted from the wild-type, ΔoxyR::Km, ΔsoxR::Km, ΔoxyR::Km-omp33::TOPO, and ΔsoxR::Km-omp33::TOPO PARP inhibitor strains showing the lack of oxyR and soxR transcription in the corresponding mutants. The gyrB gene was used as a housekeeping gene. The lengths of cDNAs obtained are indicated. Construction of double knockout mutants With the purpose of generating double knockout mutants, the recombinant plasmid pTOPO33int was transformed into both ΔoxyR::Km and ΔsoxR::Km mutants. After selection on zeocin- and kanamycin-containing plates, the ΔoxyR::Km-omp33::TOPO and ΔsoxR::Km-omp33::TOPO A. baumannii double knockout mutants were obtained. PCR tests with locus-specific primers revealed that both mutants had

fragments of the expected size (data not shown). In addition, gene disruption in mutant clones was further confirmed by sequencing the PCR products obtained, by transcriptional analyses to detect the oxyR and soxR genes (Figure 5), and by Western blot analyses selleck products to detect the omp33 gene (data not shown). Discussion Allelic mutation experiments enable investigation of the functions of many unknown genes identified during the sequencing of entire

genomes. A number of methods can be used to inactivate bacterial chromosomal genes. As mentioned above, disruption of the A. baumannii chromosome can be achieved by integration of a plasmid into the chromosome by single crossover recombination [10]. For this purpose, an internal fragment that is homologous to the target gene must be cloned into a non-replicating plasmid carrying at least one antibiotic resistance cassette. However, the stability of this Ribonucleotide reductase type of mutant must be taken into account, because if the gene-disrupted mutant cells

are grown in a medium lacking antibiotic pressure, the integrated sequence could be removed, and the disrupted gene could revert to the original wild-type [16]. We tested this possibility, and found that this is indeed the case, as also found in similar studies with E. coli [16]. Therefore, one limitation of the method is that the resulting mutants should always be maintained in an appropriate medium containing selective antibiotics. Another disadvantage of the method is that further manipulations of the mutant strain are restricted, because the same vector cannot be used (because undesired recombination events would be highly likely), thus making it impossible to construct multiple gene knockout mutants. The gene replacement method has recently been used to generate stable A. baumannii mutants [11–13]. This method is based on integration of a plasmid containing the inactivated gene of interest into the bacterial chromosome by single crossover recombination, followed by resolution (or excision) of the integrated DNA by a second recombination event, resulting in replacement of the original wild-type gene by the inactivated gene. The key step in this procedure, in A.

Data (mean ± standard deviation) of two independent experiments are presented. (PDF 5 KB) Additional file 3: Description of subpopulation “”Dead”". P. putida wild-type (A, C, E) and colR-deficient (B, D, F) strains were grown for 24 h on glucose minimal plates supplemented with 3 mM phenol. Cells were stained

with SYTO9 alone (A, B) or with SYTO9 and PI (C-F) and analysed by flow cytometry. Fluorescence at 530 (30) is plotted against fluorescence at 616 (23) nm (A-D) or side scatter of light (SSC-A) (E, F). Fluorescence at 530 (30) measures SYTO9 fluorescence and side scatter of light correlates with size of bacterial cells. (PDF 29 KB) References 1. Dominguez-Cuevas P, Gonzalez-Pastor JE, Marques S, Ramos JL, de Lorenzo V: Transcriptional Tradeoff between Metabolic and Stress-response 3-MA chemical structure Programs RG7204 ic50 in Pseudomonas putida KT2440 Cells Exposed to Toluene. J Biol Chem 2006,281(17):11981–11991.PubMedCrossRef 2. Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-Gonzalez MI, Rojas A, Teran W, Segura A: Mechanisms of solvent tolerance

in gram-negative bacteria. Annu Rev Microbiol 2002, 56:743–768.PubMedCrossRef 3. Sikkema J, de Bont JA, Poolman B: Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 1995,59(2):201–222.PubMed 4. Hallsworth JE, Heim S, Timmis KN: Chaotropic solutes cause water stress in Pseudomonas putida . Environ Microbiol 2003,5(12):1270–1280.PubMedCrossRef 5. Wery J, de Bont JAM: Solvent-tolerance of Pseudomonads: a new degree of freedom in biocatalysis. In Pseudomonas: Biosynthesis of macromolecules

and molecular metabolism. Volume 3. Edited by: Ramos JL. New York: Kluwer Academic/Plenum Publishers; 2004:609–634. 6. Hoch JA, Varughese KI: Keeping signals straight in phosphorelay signal transduction. J Bacteriol 2001,183(17):4941–4949.PubMedCrossRef 7. Dekkers LC, Bloemendaal CJ, de Weger LA, Wijffelman CA, Spaink HP, Lugtenberg BJ: A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365. Mol Plant Microbe Interact 1998,11(1):45–56.PubMedCrossRef 8. Kivistik PA, Histone demethylase Putrinš M, Püvi K, Ilves H, Kivisaar M, Hõrak R: The ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol. J Bacteriol 2006,188(23):8109–8117.PubMedCrossRef 9. Hõrak R, Ilves H, Pruunsild P, Kuljus M, Kivisaar M: The ColR-ColS two-component signal transduction system is involved in regulation of Tn 4652 transposition in Pseudomonas putida under starvation conditions. Mol Microbiol 2004,54(3):795–807.PubMedCrossRef 10. Putrinš M, Ilves H, Kivisaar M, Hõrak R: ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida . Environ Microbiol 2008,10(10):2886–2893.PubMedCrossRef 11.